Dre-Cre 顺序重组为在小鼠中标记和操作视网膜神经节细胞提供了新工具。
Dre - Cre sequential recombination provides new tools for retinal ganglion cell labeling and manipulation in mice.
机构信息
National Eye Institute, NIH, Bethesda, Maryland, United States of America; Biology Department, Babes-Bolyai University, Cluj-Napoca, Cluj, Romania.
National Eye Institute, NIH, Bethesda, Maryland, United States of America.
出版信息
PLoS One. 2014 Mar 7;9(3):e91435. doi: 10.1371/journal.pone.0091435. eCollection 2014.
BACKGROUND
Genetic targeting methods have greatly advanced our understanding of many of the 20 Retinal Ganglion Cell (RGC) types conveying visual information from the eyes to the brain. However, the complexity and partial overlap of gene expression patterns in RGCs call for genetic intersectional or sparse labeling strategies. Loci carrying the Cre recombinase in conjunction with conditional knock-out, reporter or other genetic tools can be used for targeted cell type ablation and functional manipulation of specific cell populations. The three members of the Pou4f family of transcription factors, Brn3a, Brn3b and Brn3c, expressed early during RGC development and in combinatorial pattern amongst RGC types are excellent candidates for such gene manipulations.
METHODS AND FINDINGS
We generated conditional Cre knock-in alleles at the Brn3a and Brn3b loci, Brn3a(CKOCre) and Brn3b(CKOCre). When crossed to mice expressing the Dre recombinase, the endogenous Brn3 gene expressed by Brn3a(CKOCre) or Brn3b(CKOCre) is removed and replaced with a Cre recombinase, generating Brn3a(Cre) and Brn3b(Cre) knock-in alleles. Surprisingly both Brn3a(Cre) and Brn3b(Cre) knock-in alleles induce early ubiquitous recombination, consistent with germline expression. However in later stages of development, their expression is limited to the expected endogenous pattern of the Brn3a and Brn3b genes. We use the Brn3a(Cre) and Brn3b(Cre) alleles to target a Cre dependent Adeno Associated Virus (AAV) reporter to RGCs and demonstrate its use in morphological characterization, early postnatal gene delivery and tracing the expression of Brn3 genes in RGCs.
CONCLUSIONS
Dre recombinase effectively recombines the Brn3a(CKOCre) and Brn3b(CKOCre) alleles containing its roxP target sites. Sequential Dre to Cre recombination reveals Brn3a and Brn3b expression in early mouse development. The generated Brn3a(Cre) and Brn3b(Cre) alleles are useful tools that can target exogenously delivered Cre dependent reagents to RGCs in early postnatal development, opening up a large range of potential applications.
背景
遗传靶向方法极大地促进了我们对从眼睛向大脑传递视觉信息的 20 种视网膜神经节细胞（RGC）类型的许多认识。 然而，RGC 中基因表达模式的复杂性和部分重叠要求使用遗传交叉或稀疏标记策略。 携带 Cre 重组酶的基因座与条件敲除，报告基因或其他遗传工具结合使用，可用于靶向细胞类型消融和特定细胞群体的功能操作。 转录因子 Pou4f 家族的三个成员，Brn3a，Brn3b 和 Brn3c，在 RGC 发育早期表达，并在 RGC 类型中组合表达，是进行此类基因操作的理想候选者。
方法和发现
我们在 Brn3a 和 Brn3b 基因座上生成了条件性 Cre 敲入等位基因，Brn3a（CKOCre）和 Brn3b（CKOCre）。 当与表达 Dre 重组酶的小鼠杂交时，内源 Brn3 基因由 Brn3a（CKOCre）或 Brn3b（CKOCre）表达，被删除并被 Cre 重组酶取代，产生 Brn3a（Cre）和 Brn3b（Cre）敲入等位基因。 令人惊讶的是，Brn3a（Cre）和 Brn3b（Cre）敲入等位基因均诱导早期普遍重组，与生殖系表达一致。 然而，在发育的后期，其表达仅限于 Brn3a 和 Brn3b 基因的预期内源模式。 我们使用 Brn3a（Cre）和 Brn3b（Cre）等位基因将 Cre 依赖性腺相关病毒（AAV）报告基因靶向 RGC，并证明其在形态特征，早期产后基因传递和追踪 RGC 中 Brn3 基因表达中的用途。
结论
Dre 重组酶有效地重组了包含其 roxP 靶位点的 Brn3a（CKOCre）和 Brn3b（CKOCre）等位基因。 顺序的 Dre 到 Cre 重组揭示了早期小鼠发育中的 Brn3a 和 Brn3b 表达。 生成的 Brn3a（Cre）和 Brn3b（Cre）等位基因是有用的工具，可将外源性提供的 Cre 依赖性试剂靶向早期产后发育中的 RGC，从而开辟了广泛的潜在应用领域。
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